1. Field of the Invention
The invention in the field of biochemistry and immunology medicine relates to a method of cell-free antigen processing for MHC class II-restricted antigens that exploits the role of HLA-DM in the selection of immunodominant epitopes and permits de novo identification of immunodominant epitopes in antigens, including novel antigens for which immunodominant epitopes have not yet been identified.
2. Description of the Background Art
The ability of the immune system to focus T cell responses to a select number of epitopes potentially derivable from a complex antigen is termed “immunodominance.” Several models that attempt to explain how immunodominance is established include kinetic stability for MHC binding (Lazarski, C A et al., Immunity 23:29-40 (2005)) and/or prevalence (Nelson, C A et al. Proc Natl Acad Sci USA 89:7380-3 (1992)). Other models explain immunodominance by the development of high avidity T cells recognizing those dominant peptides epitopes (Kedl, R M et al., J Exp Med 192:1105-13. (2000)).
For MHC class II restricted epitopes, HLA-DM (“DM”), a MHC-like molecule implicated in the editing of peptides that bind MHC II (benzin LK & Cresswell, P, Cell 82:155-65 (1995); Kropshofer, H. et al., EMBO J 15:6144-54 (1996)), may be involved in immunodominant epitope selection (Nanda N K et al., J Exp Med 192:781-8. (2000)). DM distinguishes its substrates (Chou C L et al., J Exp Med 192:1697-1706 (2000); Belmares, M P et al., J Immunol:169:5109-17 (2002); Pashine, A et al., Immunity 19:183-92 (2003); Stratikos, E et al., J. Immunol. 172:1109-17 (2004); Pu, Z et al. Immunity 20:467-76 (2004)) based on conformational differences between peptide/MHC complexes (Sadegh-Nasseri, S et al., Nature 353:167-70 (1991); Sadegh-Nasseri, S et al., Immunol Today 13:43-6 (1992); Sadegh-Nasseri, S et al. Nature 370: 647-50 (1994); Joshi, R et al., Biochemistry 39:3751-62 (2000).
An empty or partially filled hydrophobic pocket 1 of HLA-DR1 constitutes a favorable substrate for DM interaction, whereas a compact and rigid conformation induced by the filling of pocket 1 (Sadegh-Nasseri, S et al., 1994, supra; 16. Stern L I et al., Cell 68:465-77 (1992); Natarajan, S K et al., J. Immunol. 162:3463-70 (1999)) is an unfavorable substrate (Chou et al., supra; Pu et al., supra).
A number of approaches have been utilized for identification of T cell epitopes that include peptide mapping, screening of synthetic peptide (or combinatorial) libraries, generation and screening of expression libraries derived from organisms or tissues of interest, and elution and sequencing of naturally occurring peptides from MHC molecules using high performance liquid chromatography (HPLC) coupled with mass spectrometry (MS). Peptide mapping and the screening of synthetic peptide libraries provide critical information, but may not identify naturally occurring ligands. Elution and sequencing methods have the advantage of providing direct information about peptide generation and MHC selection in vivo. These methods are useful in identifying cytotoxic T cell epitopes, but are less effective or ineffective in defining helper T cell epitopes, which is one of the goals of the present invention.
Examples of prior art methods for screening synthetic peptides for defining helper T cell epitopes include the following. One method is based on scanning of non-overlapping short peptides of 10-15 amino acids to encompass the entire length of a given protein. T cells from immunized individuals are the common “read-out” system for identification of the immunogenic epitopes. A major problem here is that the actual immunogenic peptide sequences may go undetected because the active region of the immunogenic peptides might not fall within the sequences chosen.
In a second approach designed to circumvent this problem, peptide sequences are designed that encompass the length of a given protein with 12-15 residue peptides of overlapping sequences. This approach although preferred, is even more tedious, costly, and often unreliable. Because of the huge number of peptides that need to be tested, and limitations on the responding T cell sources, characterization of individual peptides becomes an impossible task.
A third approach employs prediction algorithms based on MHC class II structure and the nature of eluted peptides from a given MHC protein. However, this approach relies on prediction of peptides that might bind MHC II stably but might not necessarily be immunogenic; hence this method is most unreliable.
Thus, all currently established and commonly practiced methods for identifying immunogenic epitopes are fraught with disadvantages including the fact that they are very tedious, time consuming, costly, and frequently unsuccessful. Consequently, identification of a high throughput method to isolate most likely candidate immunogenic and immunodominant epitopes derived from the natural processing of protein antigens is a most desirable goal to which the present invention is directed.
Citation of the above documents is not intended as an admission that any of the foregoing is pertinent prior art. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicant and does not constitute any admission as to the correctness of the dates or contents of these documents.